Historically, nearly all efforts to achieve efficient protein binding to, for example, polystyrene surfaces by passive, noncovalent adsorption have employed the very basic carbonate or mixed bicarbonate/carbonate buffers at pH 9.6 as CB. See, for example, Butler, J. E. et al., J. Immunological Methods, 150: 77-90 (1992). This type of buffer has provided good results when used with classical first-generation hydrophobic assay plates or second-generation hydrophilic assay plates, but not when used with TC-treated plates. If an alternative buffer is employed, it is most commonly phosphate buffer, phosphate-buffered saline or tris buffer at pH of 7.0-8.0. In general, the use of organic CB's other than tris in protein immobilization has been ignored. Certainly, the use of organic CB's in conjunction with TC-treated plates for the purpose of efficient protein immobilization has been ignored, since TC-treated plates have not generally been used to perform heterogeneous immunoassays.
The phenomenon of passive noncovalent binding of protein species to polystyrene is well-known as a practical means of immobilization of assay components, where such immobilization is useful to allow rapid, simple and efficient "bound vs. free" separation(s) to be performed in support of specific detection of particular analyte(s). In general, solid phase binding-based (i.e., heterogeneous) assay formats employ immobilized species ("Capture Antibody" [CAb], if it is an antibody [Ab]) as the starting point for building an appropriate signal-mediating specific binding cascade on the solid support surface. This binding cascade is engineered such that the presence of analyte in the test sample is either: a) required in order to complete the binding cascade, as a necessary prerequisite before signal production may occur ("direct assay" format); or, b) required in order to inhibit binding of a detectable "conjugate" species, which consists of a chemically derivatized version of the analyte (indirect or "inhibition assay" format). The label/reporter species of the conjugate must include functionality which supports detection by appropriate means (e.g., bearing radioisotope, fluorophore or enzyme "label" and/or "reporter" species), while retaining essential binding motifs of the analyte fragment which are required for specific interaction with the CAb species and/or other binding partner(s). With the direct assay format, signal produced in the assay is proportional to the amount of analyte present in the sample. Alternatively in the inhibition assay format, the signal produced by the binding cascade is inversely proportional to the analyte concentration, due to the competition for a limited number of binding cascade sites between analyte (variable amount; detection not facilitated) and conjugate (fixed amount added per tube or well; detectable by design).
In many cases, the protein to be immobilized consists of an antibody (such as, for example, immunoglobulin G [IgG]) which exhibits specific binding with high affinity to the analyte of interest. In other cases, non-antibody proteins which exhibit specific binding capability (e.g., streptavidin, which is known to bind biotin with extremely high specificity and affinity, K.sub.eq= 10.sup.15 M.sup.-1) may be immobilized. In general, to be of practical utility, such immobilization needs to exhibit the following properties: a) high efficiency of protein binding (high level of polystyrene surface coverage [about 100-400 ng/cm.sup.2 ], ideally with a high fraction of input protein bound [e.g., 10-99%]); b) high stability of immobilized protein with respect to [undesired] wash-off during "bound vs. free" separation ("wash") steps; c) high retention of native conformation and biological activity; as well as d) high, substantially complete retention of binding properties of the immobilized protein vs. its solution-phase counterpart (in terms of binding affinity, binding specificity and kinetic parameters). Finally, the immobilization process must not introduce conformational or other changes in the CAb or other immobilized species which result in "non-specific binding interactions"(NSB) with other assay reagents and/or sample components. Since in general a large portion of the immobilized CAb (typically about 90% for polyclonal antibodies [pAb's], 90-99% for monoclonal antibodies [mAb's]) or other first binding partner is denatured in the course of immobilization, the latter concern regarding possible NSB is not a trivial one.
The literature, such as Butler et al., supra, indicates that passive adsorption of proteins on polystyrene is an extremely complex, incompletely understood and often unpredictable phenomenon. Historically, assay plate manufacturers have dealt with this serendipitous aspect of the application arena by providing a family of assay plate products which provide a range of polystyrene surface characteristics, from hydrophobic to hydrophilic in character. By screening a variety of tailored surface chemistries for their ability to support efficient immobilization of the desired CAb or other first binding partner, an appropriate solid phase surface chemistry can be selected which allows adequate assay performance to be demonstrated. While it is generally understood that varying the pH of the "Coating Buffer"(CB) used (i.e., the CAb diluent) can modulate the binding obtained, for theoretical reasons primarily related to the presence of a "Linear Binding Region"(LBR) in "% bound" plots of CAb binding as a function of amount of input CAb when using pH 9.6 buffer only as the CB, the vast majority of passive adsorption experiments have employed the carbonate or carbonate/bicarbonate buffer systems at pH 9.6 as "standard CB". The rationale offered for this observation was that the efficiency of passive adsorption is dependent on aggregation of the protein to be immobilized, and that such aggregation was disfavored at pH values more acidic than that of the "standard pH 9.6 CB" when Ab concentration is low. Accordingly, most skilled practitioners of the art employ pH 9.6 buffer(s) (or variants thereof) as CB exclusively, and simply test a large number of possible assay plate types in conjunction with the "standard pH 9.6 CB" to optimize CAb or first binding partner immobilization in their assay development efforts.
It is important to note that early assay plates were either underivatized or lightly derivatized (e.g., gamma irradiated) polystyrene materials of predominately hydrophobic nature. Later developments in "high protein binding" plates provided considerably more hydrophilic surfaces, e.g. by UV-irradiation of the polystyrene surface. The hydrophilic character of the second-generation assay plates proved to be superior in most cases of protein immobilization, since in general proteins contain a significant density of polar functional groups on their surface. However even using these more polar surfaces, it was evidently still advantageous to work at basic pH to avoid excessive charge density on the protein surface. At pH 9.6, the major difference relative to physiological pH or other more acidic conditions is that free amino groups of the protein are typically partially or completely deprotonated (in free base form, thus neutral with no charge) instead of fully protonated (in positively charged, ammonium ion form).
In parallel to the above developments, a different class of "assay plate" was developed for the purpose of supporting mammalian cell attachment and growth ("tissue culture" [TC] plates). TC plates are prepared using a high energy plasma treatment process under oxidative conditions, either performed under partial vacuum as is done to make Falcon.RTM. standard TC and Primaria.RTM. TC plates, or alternatively at atmospheric pressure (corona discharge process). These TC plates exhibit a high degree of surface oxidation, and in retrospect it appears that there may be a higher ratio of carboxylate groups present vs. hydroxyl groups, than is the case with classical high protein binding assay plates. Some practitioners do carry out (non-binding based) homogeneous assays in TC plates due to the superior wettability of TC-treated polystyrene plates. Also, some assay developers may indeed have conducted heterogeneous assays with TC-treated plates (e.g., inhibition assays where a high amount of immobilized CAb is not required or desirable). However, the prior literature has not taught knowledgeable practitioners of the art how to effectively employ TC-treated plates as high-protein-binding-capable assay plates on a par in performance with the classical high-protein-binding assay plates. This is accomplished by the methods and materials of the present invention.